Efficient methods and compositions for multiplex target amplification PCR

ABSTRACT

The present disclosure relates to methods of enzymatic treatment of double-stranded PCR amplified products for eliminating or minimizing primer-dimers in multiplex PCR reactions and for the efficient ligation of adapters. The present disclosure relates to methods and compositions that allow more efficient highly multiplex target amplification compared to conventional methods, compositions and kits by minimizing laboratory steps, eliminating primer-dimers and increasing the efficiency of adapter ligation. The disclosed methods use multiple target-specific primers for specific and selective amplification of targets in a subject&#39;s genome. The disclosed methods can be used for numerous downstream procedures and analysis, including DNA sequencing.

BACKGROUND OF THE INVENTION

Rapid and accurate identification of genetic variants contributing todisease, drug response or adverse drug effects is crucial for thediagnosis, individualized treatment, companion diagnostics and prognosisof patients. Many cancers and congenital or inherited disorders arecomplex diseases, which may be linked to multiple genes and may involveheterozygous mutations. Moreover, these mutations may exist in a smallquantity in a given sample.

Targeted gene sequencing is an effective approach for analyzing specificmutations and genetic changes in a sample. Target gene sequencing allowsa focus deeper upon a set of selected genes or gene regions that areknown, or suspected, to associate with a particular disease. Multiplegenes can be analyzed in parallel across the genome, thereby reducingtime and cost. Targeted gene sequencing also generates a more detaileddata set for the genes of interest, providing high sensitivity,specificity and in-depth coverage, allowing for easier gene analysis andthe detection of rare gene variants.

Moreover, because the majority of known disease-causing mutations occurin the coding region of genes, concentrating the focus for a sequenceanalysis on a panel of genes relevant to a particular disease is morepractical. Targeting, capturing, and sequencing the genomic regions ofinterest is especially valuable in clinical settings, so that eachtarget can be analyzed with greater depth and large number of specificgene panels and high sample numbers can be analyzed simultaneously. Assuch, there remains a great need to design applications where a focus onspecific genomic intervals or gene sets is required.

While whole genome sequencing is becoming more cost-effective and morepractical for many indications, a focused target-specific panelcontinues to offer the advantages of better coverage of targeted regionsand a greater ability to detect multiple variant types (including CNVsand complicated genomic rearrangements) at substantially lower costs,higher throughput, simpler bioinformatics analysis, and more focusedtesting. Such focused target-specific panels obviate the need to dealwith the secondary/incidental findings that otherwise inevitably arisewith whole genome sequencing. Furthermore, targeted sequencing ofspecific regions of interest in a large number of samples is much morecost-effective in characterizing a disease state than sequencing thewhole genomes of fewer individuals. Wider coverage of specific targetsand deeper sequencing of enriched targets allows a broader dynamic rangein allele frequencies and detection of minority sequences and lowfrequency variations for disease evaluation, treatment and prognosis. Anefficient and specific target enrichment method allows more efficienttargeted sequencing. The important parameters for target enrichment are:(i) sensitivity; (ii) specificity; (iii) uniformity; (iv)reproducibility; (v) cost; (vi) ease of use; and (vii) the amount of DNArequired per experiment.

The human genome contains approximately 3 billion bases, about 21,000coding genes and over 220,000 exons. The exons represent around 1-2% ofthe genome, and there are nine exons per gene on an average scale withan average exon size of 170 nucleotides. Next generation sequencing(NGS) is an important tool for analyzing the genome, has a highersensitivity than Sanger sequencing, allowing the detection of mutationsfrom a sample containing just a few cells. It can be utilized to detectmultiple sequence variations such as single and multi-nucleotidevariants, insertions, deletions and gene copy number variations in DNAand RNA. NGS can also be utilized to analyze gene expression levels bymeasuring quantitatively the levels of mRNA, microRNA and factorsaffecting them like gene promoter methylation. Currently there are threetypes of NGS for sequencing DNA: (a) whole-genome sequencing (WGS); (b)whole-exome sequencing (WES); and (c) targeted sequencing. WGS coversand analyzes the entire genomic content of an individual, while WEScovers only the protein-coding regions of the genome. Targetedsequencing, in contrast, focuses on a set of genes or specific regionsof the genome that are linked by common pathological mechanisms or knownclinical phenotype.

Remarkably, NGS is revolutionizing molecular characterization ofinherited diseases and cancers both for discovery of driver mutationsand the routine screening of genomic aberrations. Because of a widerange of different applications, NGS has covered many fields of lifesciences and is significantly impacting medical genetics, both inresearch and diagnostics. Isolating high-priority segments of thegenomes immensely enhances the outcome in clinical, diagnostic andresearch settings. Using NGS to focus on particular regions of interest,however, requires enrichment of relevant target regions. Notably, targetenrichment allows increased coverage of target regions and specificregions of interest, facilitating multiplexing of samples andsimplifying sequence read data analysis. Fundamental advantages oftarget enrichment in genomic assays include enrichment factor, coverageor read depth, uniformity or evenness of coverage across the targetregion of interest, reproducibility, specificity that ison-target/off-target ratio of sequence reads, required input DNA amountand overall cost per target base of useful sequence data.

PCR-based target enrichment methods are relatively fast, require fewsteps and little input DNA, and as a result are more suitable forsamples containing low amounts of input DNA, such as FFPE, cfDNA andctDNA. The specificity of PCR for target enrichment of regions ofinterest is significantly influenced by the number of primers in thereaction and primer characteristics such as G-C content and presence ofvariations in target regions might interfere with optimal primerhybridization, causing amplification failure of certain sequences alsoknown as allele drop-out. Amplicon size and coverage are importantconsiderations for PCR based target enrichment to generate even anduniform coverage.

Multiplex amplification of target nucleic acid sequences allows a greatnumber of applications in a single polymerase chain (PCR) reaction. Theadvantage of using multiple target-specific primers in a single PCRreaction is that doing so allows multiplex amplification of selectivetargets in an efficient manner, saving time, lowering cost and reducinglabor as well as increasing throughput. However, increasing the numberof oligonucleotide primers in a reaction may introduce primercross-reactivity and the formation of amplification artifacts such asprimer-dimers or non-specific priming leading to non-specificamplification products. Furthermore, some nucleic acid targets may notbe amplified due to this cross-reactivity, causing nucleic acid targetdropouts. These amplification artifacts may overly consume amplificationcomponents and reagents such as dNTPs and DNA polymerase, affecting theoverall efficiency and quality of the amplification reaction. Theseartifacts from highly multiplex amplifications may also affect thedownstream procedures such as sample preparation for next-generationsequencing. In such circumstances, the amplification of non-specificamplifications or artifacts can be carried to the downstream steps suchas next-generation sequencing read results, generating overly dominantnon-informative sequencing reads.

Multiplex amplification can amplify multiple targets of interest in onereaction and advantageously increase the number of target regions thatcan be amplified in a single reaction starting from limited amounts ofDNA where hundreds to thousands of target regions can be amplifiedsimultaneously for sequencing. Selective multiplex amplification has awide range of applications in clinical and research settings and can beused for mutation detection and analysis, single nucleotidepolymorphisms (SNPs), microbial and viral detection, deletions andinsertions, genotyping, copy number variations (CNV), epigenetic andmethylation analysis, gene expression, and transcriptome analysis. Theseapplications can be used for diagnostics, prognosis and the treatment ofdisease.

As the number of nucleic acid target regions for selective amplificationincrease, however, proportionally more primers need to be introducedinto the reaction. Higher primer numbers and concentrations in a singletest reaction may increase the amplification artifacts such asprimer-dimers, non-specific amplifications, super-amplicons, and cancause failed amplification due to interference between primers, each ofwhich can negatively impact downstream steps. A common approach to avoidor minimize these amplification artifacts is the use of commercial orin-house software packages to design primers for multiplex amplificationassays to avoid or lower the chance of primer-dimer formations andnon-specific priming. This can be done by: (1) using stringent designconsiderations to design target specific primers to mitigate primerinteractions; and (2) grouping primers into optimal subsets ofnon-overlapping pools to avoid artifacts.

Such efforts, however, do not completely solve the aforementioned issuesand, as such, there is a great need for methods or compositions forhighly multiplex amplification of target-specific sequences without orminimal amplification artifacts such as primer-dimers and non-specificamplification products as well as eliminating or minimizing primergrouping for separate test reactions, which adds extra steps.

SUMMARY OF THE INVENTION

In some embodiments, the present disclosure relates to methods,compositions and kits for multiplex target amplification and targetenrichment prior to downstream analysis such as next-generationsequencing. In certain embodiments, the present disclosure relates to amethod comprising the step of using a plurality of target-specificprimers to perform target enrichment amplification in a DNA or RNAsample, wherein the amplification is conducted under optimal conditionsin the presence of amplification reagents comprising polymerase anddNTPs. In various embodiments, the method further comprises the step ofconverting mRNA in a sample to cDNA. In some embodiments, thetarget-specific primers of the method comprise a target-specificsequence at the 3′-end and an auxiliary sequence at the 5′-end, whereinthe auxiliary sequence is configured to allow digestion of PCR productaround the methylated site with a proper restriction enzyme.

In some embodiments, the disclosure is directed to a method comprisingthe steps of: (1) in a test reaction, hybridizing two or moretarget-specific primers to nucleic acid target sequences, wherein thetarget-specific primers comprise a methylated universal auxiliaryportion with a methylation-dependent endonuclease restriction enzymerecognition site and a target-specific portion configured to targetnucleic acid target sequences in the sample; (2) subjecting the testreaction to amplification under optimal amplification conditions toproduce an amplified product comprising an amplicon; (3) subjecting theamplified product to digestion with a methylation-dependent endonucleaserestriction enzyme to form a digestion product, wherein the digestionproduct comprises amplicons comprising sticky ends (i.e., unpairednucleotides) on each end of the strands; (4) performing size selectionpurification on the digestion product for removal of digestedprimer-dimers and unused primers to form digested amplicons comprisingdsDNA; (5) ligating universal adapters to dsDNA from the digestedamplicons to form a ligation product, wherein the ligation universaladapters comprise a universal sequence portion and sticky ends; (6)subjecting the ligation product to amplification with barcoded universalprimers complementary to a sequence on the ligating universal adaptersto form a final amplification product; and (7) quantifying the finalamplification product for next-generation sequencing.

In some embodiments, the disclosure is directed to a method comprisingthe steps of: (1) in a test reaction, hybridizing two or moretarget-specific primers to nucleic acid target sequences, wherein thetarget-specific primers comprise a complementary universal auxiliaryportion at the 5′-end and a target-specific portion configured to targetthe nucleic acid target sequences in the sample; (2) subjecting the testreaction to a first amplification by universal auxiliary primers underoptimal amplification conditions to form an amplified product; (3)subjecting a portion of the amplified product to a second amplificationusing a methylated universal auxiliary primer to form a second amplifiedproduct, wherein the methylated universal auxiliary primer comprises arestriction enzyme recognition sequence; (4) subjecting the secondamplified product to digestion with a methylation-dependent endonucleaserestriction enzyme to form a digestion product comprising ampliconscomprising sticky ends on each end of the strands; (5) performing sizeselection purification on the digestion product to remove digestedprimer-dimers and unused primers to form digested amplicons comprisingdsDNA; (6) ligating universal adapters to dsDNA from the digestedamplicons to form a ligated product, wherein the ligating universaladaptors comprise complementary sticky ends and a universal sequenceportion; (7) subjecting the ligated product to a third amplificationusing barcoded universal primers complementary to sequences on theligating universal adapters; and (8) quantifying the final amplificationproduct for next-generation sequencing.

In some embodiments, the disclosed methods comprise the steps of: (1)annealing at least 10, 20, 100, 500, 1,000, 2,500, 5,000, 10,000,25,000, 50,000, 80,000, 100,000, 150,000 or greater target nucleic acidsequences to target-specific primers, wherein the target-specificprimers comprise both forward and reverse primers; and (2) generatingtarget amplification products using primer extension, wherein the targetamplification products comprise different sized amplicons. In someembodiments, the disclosed methods further comprise the step ofdetermining the presence or absence of at least one target amplificationproduct. In some embodiments, the disclosed methods further comprise thestep of determining the sequence of at least one target amplificationproduct. In some embodiments, the disclosed methods comprise the step ofusing at least 10, 20, 100, 500, 1,000, 2,500, 5,000, 10,000, 25,000,50,000, 80,000, 100,000, 150,000 or more forward primers and reverseprimers, wherein each forward primer and reverse primer is directed tohybridize to a specific target nucleic acid sequence. In certainembodiments, the disclosed methods further comprise the step ofperforming RNA analysis for RNA expression measurements both in acontrol sample and a subject sample by comparison of control expressionlevel to the subject sample expression level.

In certain embodiments of the disclosed methods, amplification isperformed using state-of-art polymerase chain reaction (PCR). In certainembodiments, the performance of PCR comprises an annealing time greaterthan 0.1, 0.5, 1, 2, 5, 8, 10 or 15 minutes. In certain embodiments, theperformance of PCR comprises an extension time greater than 0.1, 0.5, 1,2, 5, 8, 10 or 15 minutes.

In some embodiments, the disclosed methods further comprise the stepsof: introducing modifications to amplified products wherein themodifications enable digestion by endonucleases; utilizing restrictionendonucleases to digest the amplification products, wherein restrictionendonucleases are configured to cleave DNA in the amplification productat a fixed distance away from the modifications. In some embodiments,the modification is a methylation. In some embodiments, the methylatedamplified products are digested at specified restriction sites bymethylation-dependent endonuclease restriction enzymes. In someembodiments, the methylation-dependent endonuclease restriction enzymesare configured to digest double-stranded DNA. In some embodiments, themethylation-dependent endonuclease restriction enzymes are configured todigest both double-stranded DNA and single-stranded DNA.

In some embodiments of the method, the target specific primers are at aconcentration of 500, 250, 100, 80, 70, 50, 30, 10, 2, or 1 nM. In someembodiments, the GC content of the target-specific primers is between40% to 70%, or 30% to 60%, or 50% to 80%. In some embodiments, thetarget-specific primer GC content range is less 20%, 15%, 10% or 5%. Insome embodiments, the melting temperature (T_(m)) of the target-specificprimers is between 55° C. to 65° C., 40° C. to 70° C., or 55° C. to 68°C. In some embodiments, the length of the target-specific primers isbetween 20 and 90 bases, 40 and 70 bases, 20 and 40 bases, or 25 and 50bases. In some embodiments, the 5′-region of the modified methylatedprimer comprises an auxiliary sequence that is not complementary orspecific for any nucleic acid region in the sample. In some embodiments,the target specific primers anneal specifically to different regions ona target region, gene or different exons of a gene. In certainembodiments, the target-specific primers are configured to anneal totarget regions and simultaneously amplify the target nucleic acids inthe sample.

In some embodiments, the present disclosure is directed to a method fordetecting cancer in a sample. In some embodiments, the presentdisclosure is directed to a method for detecting the presence or absenceof a congenital or inherited disease in a sample. In some embodiments,the present disclosure is directed to a method for detecting thechromosomal ploidy status in a gestating fetus in a sample, wherein themethod comprises the step of measuring allele counts at polymorphicsites to determine the chromosomal ploidy state. In some embodiments,the method further comprises the step of selecting a treatment for asubject based on the target sequences detected in a sample.

In some embodiments, the target sequences comprise clinically actionablemutations. In some embodiments, the target sequences are associated withdrug resistance or pharmacogenetic drug (companion diagnostic)treatment. In some embodiments, detection, identification and/orquantitation of genetic markers can be related with organtransplantation or organ rejection.

In some embodiments, the sample is obtained from a healthy subject. Insome embodiments, the sample is obtained from a pregnant subject,wherein the sample comprises maternal and fetal nucleic acids. Incertain embodiments, the sample is obtained from a subject that issuspected to have a disease or an elevated chance to have the diseaseand wherein the target sequence comprises mutations or variations thatare associated with the disease. In some embodiments, the disease iscancer. In some embodiments, the sample comprises whole genomic DNA,mechanically or enzymatically fragmented DNA, cDNA, formalin-fixedparaffin-embedded tissue (FFPE), cell-free DNA (cfDNA) or circulatingtumor DNA (ctDNA). In some embodiments, the sample comprises cell-freeDNA from the blood plasma of a pregnant woman. In some embodiments,nucleic acid target sequences in a sample comprise single nucleotidepolymorphisms (SNPs), mutations, gene rearrangements and fusions, shorttandem repeats, genes, exons, coding regions and exomes. In someembodiments, the sample comprises mRNA. In some embodiments, mRNA in thesample is subjected to reverse transcription reaction to generate DNA.In some embodiments, the nucleic acids are obtained from a single cell.In some embodiments, the sample comprises nucleic acids obtained fromblood, serum, plasma, spinal fluid, urine, tissue, saliva, biopsies,sputum, swabs, surgical resections, cervical swabs, tears, tumor tissue,fine needle aspiration (FNA), circulating cell-free DNA (cfDNA), andcirculating tumor DNA (ctDNA), scrapings, swabs, mucus, urine, semen,hair, other non-restricting clinical or laboratory obtained samples or aforensic sample.

In one embodiment, at least one ligation adapter is a double strandedoligonucleotide comprising a sticky end and a universal priming sequencewherein the sticky end sequence of the ligation adapter is configured tobe complementary to one sticky end of the digestion product produced bythe methylation-dependent endonuclease restriction enzyme, wherein theuniversal priming sequence is used for downstream amplification andsequencing. In some embodiments, the disclosed method comprises the stepof ligating at least one ligation adapter to target nucleic acids. Insome embodiments, the ligation adapters comprise: (a) a universalpriming sequence; (b) a barcode sequence (a DNA tagging sequence); and(c) a sticky end wherein the sticky end sequence of the ligation adapteris complementary to the sticky ends of the digestion product produced bythe methylation-dependent endonuclease restriction enzyme. In someembodiments, the ligation adapter comprises: (a) a universal primingsequence; and (b) a sticky end, wherein the sticky end sequence iscomplementary to the digested amplicon sticky end according to themethylation-dependent endonuclease restriction enzyme used. In someembodiments, the ligation adapters comprise sticky ends complementaryfor both ends of the restriction enzyme digested product. In anotherembodiment, the ligation adapter comprises a universal priming sequenceconfigured to allow amplification of target sequences with ligatedadapter to be re-amplified. In some embodiments, the ligation step usesmore than one ligation adapter.

In some embodiments, the present disclosure is directed to a methodcomprising the steps of: contacting target-specific primers with nucleicacids in a sample, wherein the sample comprises both maternal and fetalDNA and wherein the target-specific primers are configured tosimultaneously hybridize to at least 10, 20, 100, 500, 1,000, 2,500,5,000, 10,000, 25,000, 50,000, 80,000, 100,000 or 150,000 differenttarget regions in nucleic acids; amplifying a plurality of targetnucleic acids to produce amplicons; subjecting the amplicons tonext-generation sequencing to generate sequence data; and analyzing thesequence data by a software algorithm. In some embodiments, the methodfurther comprises the step of counting the amplicons related to thecontrol chromosome and the suspected chromosome to determine thepresence or absence of an abnormal chromosome distribution. In someembodiments, the method further comprises the step of counting theamplicons to determine presence of aneuploidy in the fetal DNA. In someembodiments, the method further comprises the step of counting theamplicons and determining the presence or absence of a deletion in thenucleic acids. In some embodiments, the sample comprises both maternaland fetal DNA. In some embodiments, the sample comprises cell-free DNAfrom blood plasma of a subject.

In some embodiments, the method further comprises determining thepresence of aneuploidy or abnormal chromosome distribution by comparingthe sample to a control sample comprising control sequences. In someembodiments, the relative quantity of the control sequences is known. Insome embodiments, the relative quantity of each of the target nucleicacid sequences are standardized by a reference genome. In someembodiments, the control or reference genome includes at least onechromosomal abnormal distribution or aneuploidy. In some embodiments,the aneuploidy is at chromosome 13 or chromosome 18 or chromosome 21 orchromosome X or chromosome Y.

In some embodiments, the present disclosure is directed to a kitcomprising two or more modified methylated target-specific primersconfigured to amplify target sequences of interest in a sample. In someembodiments, the present disclosure is directed to a kit comprising twoor more modified methylated universal primers configured to amplifytarget sequences of interest in a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure can be better understood with reference to the followingdrawings. The elements of the drawings are not necessarily to scalerelative to each other, emphasis instead being placed upon clearlyillustrating the principles of the disclosure. Furthermore, likereference numerals designate corresponding parts throughout the severalviews.

FIG. 1 illustrates a schematic image of forward and reverse methylatedtarget-specific primers. Each primer consists of a target-specificsequence portion, and a universal auxiliary sequence portion consistingof a methylated nucleotide C (mC), a restriction enzyme recognition siteand a cleavage site.

FIG. 2 illustrates a schematic diagram of forward and reverse universalauxiliary methylated primer for the second approach. The universalauxiliary sequence primer consists of a methylated nucleotide C (mC), arestriction enzyme recognition site and a cleavage site.

FIG. 3 shows four examples of modification-dependent endonucleases andtheir restriction sites. The methylation-dependent endonucleaserestriction enzymes digest the modified (methylated) cytosine in thedouble-stranded DNA at the shown restriction site.

FIG. 4 illustrates an amplicon amplified with methylated primers(approach 1 or 2) and digestion by methylation/modification-dependentendonuclease enzyme. The digested primers and excess/unused primers areremoved by size selection cleanup and complementary sticky-end adaptorsare ligated to sticky-end amplicons.

FIG. 5 shows amplification of adaptor-ligated amplicons with barcodeduniversal primers prior to library preparation and sequencing.

FIG. 6 depicts a schematic drawing of the overall library preparationwherein: (1) double stranded gDNA or RNA (converted to cDNA) areamplified by either methylated target-specific primers, wherein themethylation is on the auxiliary sequence portion (approach 1); oramplification using target-specific primers that comprise a universalauxiliary sequence to form an amplified product, and then using aportion of the amplified product for the next PCR using methylateduniversal auxiliary primer (approach 2); (2) digesting either amplifiedproduct from (1) with methylation-dependent endonuclease restrictionenzyme, wherein the amplified product contains sticky ends on each endof the strands, and performing size selection purification for removalof digested primer-dimers and unused primers; (3) ligating universaladapters comprising complementary sticky ends to dsDNA wherein theligation adapter comprises a universal sequence portion; (4) subjectingthe ligated product to amplification with barcoded universal primerscomplementary to sequences on the ligation adapters to form a finalamplification product; and (5) preparing the final amplification productfor next-generation sequencing.

FIG. 7 illustrates library preparation workflow for the two methodsdisclosed by FIG. 6 .

FIG. 8 shows the bidirectional sequence results (electropherograms) formultiplex PCR with methylated primers (approach 1) for the oncogene EGFRwith the Illumina P5 adapter sequence (SEQ ID NO: 1), sequencing primer(SEQ ID NO: 2), and Illumina P7 adapter sequence (SEQ ID NO: 3) and areverse complement sequence with the Illumina P7 adapter sequence (SEQID NO: 4), sequencing primer (SEQ ID NO: 5), and Illumina P5 adaptersequence (SEQ ID NO: 6).

FIG. 9 shows the bidirectional sequence results (electropherograms) formultiplex PCR with methylated primers (approach 1) for the oncogene TP53with the Illumina P5 adapter sequence (SEQ ID NO: 1), sequencing primer(SEQ ID NO: 7), and Illumina P7 adapter sequence (SEQ ID NO: 3) and areverse complement sequence with the Illumina P7 adapter sequence (SEQID NO: 4), sequencing primer (SEQ ID NO: 8), and Illumina P5 adaptersequence (SEQ ID NO: 6).

FIG. 10 shows the bidirectional sequence results (electropherograms) formultiplex PCR with methylated primers (approach 1) for the oncogene KITwith the Illumina P5 adapter sequence (SEQ ID NO: 1), sequencing primer(SEQ ID NO: 9), and Illumina P7 adapter sequence (SEQ ID NO: 3) and areverse complement sequence with the Illumina P7 adapter sequence (SEQID NO: 4), sequencing primer (SEQ ID NO: 10), and Illumina P5 adaptersequence (SEQ ID NO: 6).

FIGS. 11A and 11B show screenshots of Illumina sequence reads fromlibrary generated with approach 2 disclosed in this invention, mappedonto human sequence reference hg19 for different targeted regions forKRAS (FIG. 11A) and KIT (FIG. 11B).

DETAILED DESCRIPTION

The present disclosure relates to methods and compositions for theamplification and enrichment of specific target sequences. The followingexamples, applications, descriptions and content are exemplary andexplanatory, and are non-limiting and non-restrictive in any way. Thepresent disclosure features a variety of applications, such asgenotyping, detection of chromosomal abnormalities (such as a fetalchromosome aneuploidy), gene mutation and polymorphism (such as singlenucleotide polymorphisms, SNPs) analysis, gene deletion, determinationof paternity, analysis of genetic differences among populations,forensic analysis, measuring predisposition to disease, quantitativeanalysis of mRNA, and detection and identification of infectious agents(such as bacteria, parasite, and viruses). The methods disclosed hereincan also be used for non-invasive prenatal testing, such as paternitytesting or the detection of fetal chromosome abnormalities.

Next-generation sequencing has enabled many applications at an extremelylow cost, but certain applications such as whole genome sequencing andwhole transcriptome sequencing, although practical for research settingsand discovery, remain impractical in clinical settings for thediagnosis, treatment and prognosis of disease. Specific and uniformmultiplex target sequencing presents many advantages in both clinicaland research settings. To increase the output and efficacy of biologicalassays, such as multiplex PCR and next-generation sequencing,simultaneous amplification of many target genes using a combination ofseveral target specific primers allows multiplex amplification ofregions of interest. Although the use of many primers reduces labor,cost and time, the resulting non-specific amplifications oramplification artifacts such as primer-primer interactions(primer-dimers) and superamplicons can interfere with optimalamplification and further analysis, such as sequencing. These artifactswaste PCR reaction reagents and generate shorter fragments in lieu ofthe intended target sequences. Further, these unwanted non-specificfragments tend to dominate the amplification reaction, because they areamplified more efficiently than the desired target sequences. Theseundesired artifacts may also interfere with downstream procedures andapplications that involve a second PCR step, such as next-generationsequencing. These artifacts may consume a sizeable portion of sequencereads, generating non-informative results.

A current problem with target enrichment—whereby genomic regions from aDNA sample are selectively captured before sequencing—is achieving highspecificity and uniformity, which would require fewer sequencing readsto generate adequate coverage and sequence data for downstream analysis.In certain applications, such as cancer or genetic diseases, much deepersequencing is needed to detect, identify or verify somatic mutationswith high specificity and uniformity in the panel.

Further, the need remains to minimize primer-dimers, which would allowfor the development of highly multiplex PCR whereby multiplexamplification could simultaneously amplify a large number of targetnucleic acids in a single test reaction. Moreover, removal ofprimer-dimers would allow an increased number of primers formultiplexing, higher concentrations of primers for balancedamplification and higher sensitivity. The ability to increase the numberof target-specific in a multiplex PCR would allow for the simultaneousamplification of a large number (thousands) of nucleic acid targetswhile also decreasing the amount of input DNA, labor and time. Thiswould be especially advantageous when the amount of starting inputnucleic acid material is limited, or the sample is nucleic acid from asingle cell.

To address the foregoing needs, herein described is a method ofmultiplex target enrichment using methylated primers to removeprimer-dimers to increase and expand the multiplex primer capability forfurther analysis such as next-generation sequencing. The method can beused in two approaches. In the first approach, the target-specificprimers contain a methylated C (mC) in the universal auxiliary portion,and in the second approach the two universal auxiliary primers containmethylated C (mC) and are used in combination with target-specificprimers containing a portion complementary to methylated universalauxiliary primer.

Thus, the present disclosure can be performed in two variant approaches.The first approach is a method comprising the steps of: (1) contactingmethylated target-specific primers with nucleic acid target sequences ina sample to hybridize the methylated target-specific primers to thetarget sequences in the sample; (2) subjecting the target nucleic acidsequences to amplification under optimal amplification conditions toform an amplified product; (3) subjecting the amplified product todigestion with a methylation-dependent endonuclease restriction enzymeto form a digestion product, wherein the digested product containssticky ends on each end of the strands; (4) performing size selectionpurification on the digestion product for removal of digestedprimer-dimers and unused primers to form a selected digestion product;(5) ligating universal adapters to dsDNA in the selected digestionproduct to form a ligated product, wherein the universal adapterscomprise complementary sticky ends to ds DNA and a universal sequenceportion; (6) subjecting the ligated product to amplification usingbarcoded universal primers to form a final amplification product,wherein the barcoded universal primers are configured to becomplementary to a sequence on the ligation adapters; and (7) preparingthe final amplification product for next-generation sequencing.

The second approach is method comprising the steps of: (1) contactingtarget-specific primers with nucleic acid target sequences in a sampleto hybridize the target-specific primers to the target sequences,wherein the target-specific primers comprise a universal auxiliaryportion at the 5′-end; (2) subjecting the target sequences to a firstamplification with universal auxiliary primers under optimalamplification conditions to form an amplified product; (3) subjecting aportion of the amplified product to a second amplification usingmethylated universal auxiliary primers to form a second amplifiedproduct, wherein the methylated universal auxiliary primers comprise arestriction enzyme recognition sequence; (4) subjecting the secondamplified product to digestion with a methylation-dependent endonucleaserestriction enzyme to form a digested product, wherein the digestedproduct contains sticky ends on each end of the strands; (5) performingsize selection purification on the digested product to remove digestedprimer-dimers and unused primers to form a selected digested product;(6) ligating universal adapters to ds DNA in the selected digestedproduct to form a ligated product, wherein the universal adapterscomprise complementary sticky ends and a universal sequence portion; (6)subjecting the ligated product to a third amplification using barcodeduniversal primers to form a final amplification product, wherein thebarcoded universal primers comprise a sequence complementary tosequences on the ligation adapters; and (7) quantifying the finalamplification product for next-generation sequencing.

The disclosed methods may further comprise the step of extracting DNAfrom a sample, such as FFPE or blood, plasma DNA or RNA (converted to dscDNA). The disclosed methods may further comprise purification known tothose of skill in the art. The disclosed methods may comprise about 10,20, 100, 500, 1,000, 2,500, 5,000, 10,000, 25,000, 50,000, 80,000,100,000 or 150,000 or greater target-specific primers and about 10, 20,100, 500, 1,000, 2,500, 5,000, 10,000, 25,000, 50,000, 80,000, 100,000or 150,000 or greater target sequences.

A subject may be a mammal. In some instances, the subject is a human.The subject may be healthy, diagnosed with a disease, or suspected ofhaving a disease. In other instances, the subject may be non-mammalian,such as a bacterium, virus, or fungus.

Target nucleic acids may be present in a sample obtained from a subject.A sample can comprise proteins, cells, fluids, biological fluids,preservatives, blood, hair, biopsy materials, and other material thatcontains nucleic acids. The nucleic acid sample may comprise genomic DNAor RNA. The sample may also comprise nucleic acid molecules obtainedfrom FFPE or archived DNA samples. The sample may also comprisemechanically or enzymatically sheared or fragmented DNA. The sample maycomprise circulating cell-free DNA (cfDNA) such as material obtainedfrom a maternal subject, or circulating tumor DNA (ctDNA) from a subjectdiagnosed with cancer or from a subject for cancer screening purposes.The sample may comprise nucleic acid molecules obtained from blood,serum, plasma, spinal fluid, urine, tissue, saliva, biopsies, sputum,swabs, formalin-fixed paraffin-embedded material (FFPE), surgicalresections, cervical swabs, tears, tumor tissue, fine needle aspiration(FNA), circulating cell-free DNA (cfDNA), and circulating tumor DNA(ctDNA), scrapings, swabs, mucus, urine, semen, hair, laser capturemicrodissections, and other non-restricting clinical or laboratoryobtained samples. The sample may be an epidemiological, bacterial,viral, fungi, agricultural, forensic or pathogenic sample.

A plurality of target-specific primers may comprise a target-specificsequence and an auxiliary sequence at the 5′-, wherein the auxiliarysequence is configured to allow digestion of a methylated site using arestriction enzyme. Further, the methylation can either be directlysituated on the auxiliary portion of a target-specific primer on theuniversal auxiliary primer. The target-specific primers may comprise amethylated universal auxiliary sequence portion that comprises arestriction enzyme recognition site and a target-specific sequenceportion (FIG. 1 ). Amplification may utilize at least one forwardtarget-specific primer and at least one reverse target-specific primer.The target-specific primers may comprise a universal auxiliary sequence.The present disclosure also contemplates a method whereintarget-specific primers, comprising a universal auxiliary portion, areused in the first amplification, and wherein complementary methylateduniversal auxiliary primers, comprising a methylated nucleotide and arestriction enzyme recognition site, are used for the secondamplification (FIG. 2 ). The target-specific primers may be configuredto hybridize to short tandem repeats (STRs) of target sequences. Thetarget-specific primers can include a nucleotide modification in the3′-end, the 5′-end, or across the sequence. The length oftarget-specific portion of the target-specific primer can be about 15 to40 bases. The T_(m) of each target-specific primer can be about 50° C.to about 72° C. or other ranges of temperature.

The target-specific primers may be directed to at least one targetsequence whereby one or more mutations in target sequence are indicativeof a subject having a disease. The mutation may be a clinicallyactionable mutation. The mutation may also be associated with drugresistance or a companion diagnostic treatment. Mutations can includesubstitutions, insertions, inversions, point mutations, deletions,mismatches and translocations. In one embodiment, the mutations caninclude variation in copy number. In one embodiment, the mutations caninclude germline or somatic mutations. In some embodiments, themutations have less than about a 10% allele frequency. In otherembodiments, the mutations have less than about a 5%, 3%, 1%, 0.5%, 0.1%or 0.01% allele frequency.

The target-specific primers may target a sequence associated withdisease related to cancer or one or more autoimmune, genetic,cardiovascular, developmental, metabolic, neurological, neuromusculardisorders, newborn diseases or newborn disorders. The target sequencemay be associated with organ transplantation or organ rejection.

The target-specific primers may be directed to one or more genesassociated with high-prevalence clinically relevant cancer genescovering many cancers. The target-specific primers may be configured toamplify one or more clinically relevant genes for cancer including, butnot limited to: AIP, ALK, APC, ATM, BAP1, BARD1, BLM, BMPR1A, BRCA1,BRCA2, BRIP1, CDH1, CDK4, CDKN1B, CDKN2A, CHEK2, DICER1, EPCAM, FANCC,FH, FLCN, GALNT12, GREM1, HOXB13, MAX, MEN1, MET, MITF, MLH1, MRE11A,MSH2, MSH6, MUTYH, NBN, NF1, NF2, PALB2, PHOX2B, PMS2, POLD1, POLE,POT1, PRKAR1A, PTCH1, PTEN, RAD50, RAD51C, RAD51D, RB1, RET, SDHA,SDHAF2, SDHB, SDHC, SDHD, SMAD4, SMARCA4, SMARCB1, SMARCE1, STK11, SUFU,TMEM127, TP53, TSC1, TSC2, VHL, and XRCC2.

The target-specific primers may also be directed to one or more genesassociated with breast cancer. The target-specific primers may beconfigured to amplify one or more clinically relevant genes for breastcancer including, but not limited to: ATM, BARD1, BRCA1, BRCA2, BRIP1,CDH1, CHEK2, FANCC, MRE11A, MUTYH, NBN, NF1, PALB2, PTEN, RAD50, RAD51C,RAD51D, STK11, and TP53.

The target-specific primers may be directed to one or more genesassociated with ovarian cancer. The target-specific primers may beconfigured to amplify one or more clinically relevant genes for ovariancancer including, but not limited to: ATM, BARD1, BRCA1, BRCA2, BRIP1,CDH1, CHEK2, DICER1, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NF1,PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, SMARCA4, STK11, and TP53.

The target-specific primers may be directed to one or more genesassociated with colorectal cancer. The target-specific primers may beconfigured to amplify one or more clinically relevant genes forcolorectal cancer including, but not limited to: APC, BMPR1A, CDH1,CHEK2, EPCAM, GREM1, MLH1, MSH2, MSH6, MUTYH, PMS2, POLD1, POLE, PTEN,SMAD4, STK11, and TP53.

The target-specific primers may be directed to one or more genesassociated with prostate cancer. The target-specific primers may beconfigured to amplify one or more clinically relevant genes for prostatecancer including, but not limited to: ATM, BRCA1, BRCA2, CHEK2, EPCAM,HOXB13, MLH1, MSH2, MSH6, NBN, PALB2, PMS2, RAD51D, and TP53.

The target-specific primers may be directed to detection andidentification of fusion genes such as abnormal gene fusions ortransforming gene fusions (example EML4-ALK or ROS1) in cancer. Thetarget-specific primers may be configured to amplify one or more fusiongenes in cancer including, but not limited to: AKT3, ALK, ARHGAP26, AXL,BRAF, BRD3, BRD4, EGFR, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, FGFR1,FGFR2, FGFR3, FGR, INSR, MAML2, MAST1, MAST2, MET, MSMB, MUSK, MYB,NOTCH1, NOTCH2, NRG1, NTRK1, NTRK2, NTRK3, NUMBL, NUTM1, PDGFRA, PDGFRB,PIK3CA, PKN1, PPARG, PRKCA, PRKCB, RAF1, RELA, RET, ROS1, RSPO2, RSPO3,TERT, TFE3, TFEB, THADA, and TMPRSS2.

The target-specific primers may be configured to amplify selectivelytarget sequences carrying mutations that are associated with acongenital or inherited disease. The mutations can be somatic orgermline mutations. Mutations associated with a congenital or inheriteddisease can include point mutations, insertions, deletions, inversions,substitutions, mismatches, translocations and copy number variations. Insome embodiments, at least one of the target-specific primers associatedwith an inherited disease is at least 90% complementary to the targetsequence.

The target-specific primers may be directed to one or more genesassociated with cardiovascular disease. The target-specific primers maybe configured to amplify one or more clinically relevant genesassociated with cardiovascular disease including, but not limited to:ABCC9, ACTA2, ACTC1, ACTN2, AKAP9, ANK2, ANKRD1, BAG3, CACNA1C,CACNA2D1, CACNB2, CALM1, CASQ2, CAV3, CBS, COL3A1, COL5A1, COL5A2,CRYAB, CSRP3, DES, DMD, DSC2, DSG2, DSP, EMD, EYA4, FBN1, FBN2, FKTN,FLNA, FXN, GATA4, GATAD1, GLA, GPD1L, HCN4, JAG1, JPH2, JUP, KCND3,KCNE1, KCNE2, KCNE3, KCNH2, KCNJ2, KCNJ5, KCNJ8, KCNQ1, LAMA4, LAMP2,LDB3, LMNA, MED12, MYBPC3, MYH11, MYH6, MYH7, MYL2, MYL3, MYLK, MYOZ2,MYPN, NEXN, NKX2-5, NOTCH1, PKP2, PLN, PLOD1, PRKAG2, PRKG1, PTPN11,RAF1, RBM20, RYR2, SCN1B, SCN2B, SCN3B, SCN4B, SCN5A, SKI, SLC2A10,SMAD3, SMAD4, SNTA1, TAZ, TBX1, TBX20, TBX5, TCAP, TGFB2, TGFB3, TGFBR1,TGFBR2, TMEM43, TMPO, TNNC1, TNNI3, TNNT2, TPM1, TRDN, TRPM4, TTN, TTR,TXNRD2, and VCL.

The present disclosure also relates to a method of target enrichment bymultiplex PCR, comprising the steps of contacting target sequences witha plurality of target-specific primers in the presence of PCR reagentssuch as DNA polymerase, dNTPs and reaction buffer; and given the optimalconditions of temperature and time for denaturation, annealing andextension, hybridizing the primers complementary target sequences andextending such target sequences. Amplification, purification and cleanupmay be adjusted or removed as needed for optimization of multiplextarget amplification for downstream processes as determined by one ofskill in the art.

The methods disclosed herein feature a broad range of applications inclinical and research settings and can be used for mutation detectionand analysis, single nucleotide polymorphisms (SNPs), microbial andviral detection, deletions and insertions, genotyping, copy numbervariations (CNV), epigenetic and methylation analysis, gene expression,transcriptome analysis, and low frequency allele mutations. Thedisclosed methods can also be used for the detection, diagnostics,prognosis and treatment of disease. The disclosed methods can alsodetect both germline or somatic mutations in a sample.

The disclosed methods may use PCR and DNA polymerase. A wide selectionof DNA polymerases are available, which feature differentcharacteristics such as thermostability, high-fidelity, processivity andHot Start. Amplification conditions, such as number of cycles, annealingtemperature, annealing duration, extension temperature and extensionduration, may be adjusted to optimal conditions for amplification, whichmay be based on the instructions provided with the commercial DNApolymerase that is selected. The concentration of DNA polymerase formultiplex PCR can be higher than for single-plex PCR.

The ligation adapters used by the methods disclosed herein aredouble-stranded and are configured to ligate to double-stranded nucleicacid fragments. The ligation adapters comprise a universal sequenceportion that is non-complementary to target sequences and a sticky endcomplementary to the sticky-end on digested amplicons. The barcodesequence on the barcoded universal primer allows tagging nucleic acidfragments of each subject and can discriminate the identity of eachsample. As such, barcoding increases throughput by enabling the poolingof samples. The disclosed methods may also use the ligation adapters forthe purpose of universal amplification of a large number of nucleic acidtarget sequences.

The disclosed methods amplify target sequences using multiplexpolymerase chain reaction, wherein more than one target sequence isamplified in a single test reaction. The amount of nucleic acid in asample needed for multiplex amplification can be about 1 ng.Alternatively, the amount of nucleic acid material can be about 5 ng, 10ng, 50 ng, 100 ng or 200 ng or more. Multiplex polymerase chain reactioncan be performed on a thermocycler and each cycle of the multiplex PCRcomprises the steps of denaturation, annealing and extension. Each cycleof the multiplex PCR includes at least one denaturation step, oneannealing step and one extension step for extension of nucleic acids.The disclosed methods can comprise 5 to 20 cycles of PCR in each roundof amplification, although other numbers of cycles are possible. Forexample, between 1 and 10 cycles, between 1 and 15 cycles, between 1 and20 cycles, between 1 and 25 cycles, or between 1 and 30 cycles or morecan be performed. Each cycle or set of cycles can have differentdurations and temperatures. For example, the annealing step can haveincremental increases and decreases in temperature and duration or theextension step can have incremental increases and decreases intemperature and duration. The duration can have decreases or increasesof about 5 seconds, 10 seconds, 30 seconds, 1 minute, 2 minutes, 4minutes, 8 minutes or greater increments. The temperature can havedecreases or increases in about 0.5, 1, 2, 4, 8, 10 Celsius or greaterincrements.

Amplicon size selection can be used to sequence amplified products of acertain length range. For example, amplicons of 100 to 250 base pairs,150 to 300 base pairs, 120 to 350 base pairs, or 200 to 500 base pairsor greater length range can be sequenced.

Typically, a small number of primers in a primer set or primer poolcause amplification artifacts such as primer-dimers in multiplexamplification reactions. By employing a primer selection algorithm thatcan calculate the undesired primer-primer interactions, however,target-specific primer selection can be performed in an efficient mannerthat minimizes primer-primer interactions to negligible amount, allowingmultiplex amplification capable of simultaneously amplifying a largenumber of target sequences in a single test reaction. Moreover, thedigestion of a methylation site on the universal portion oftarget-specific primers allows the removal of primer-dimers bymethylation-dependent endonuclease restriction enzyme digestion and sizeselection purification such as SPRIselect beads. Furthermore, methylatedtarget-specific primers allow increased number of primers formultiplexing, higher concentrations of target-specific primers forbalanced amplification and higher sensitivity without concerns forprimer-dimers. The ability to increase the number of target-specificprimers in a multiplex PCR allows the simultaneous amplification of alarge number (thousands) of nucleic acid target sequences whiledecreasing the amount of input DNA, labor and time. This is especiallyadvantageous when the amount of starting input nucleic acid material islimited, or when the sample comprises nucleic acid from a single cell.

Primer dimers can be reduced or minimized by adjusting differentparameters of the disclosed methods, such as the duration of annealingsteps, temperature increments, and/or the number of cycles of PCR. Theprimer concentrations can be lowered, and annealing temperature andduration can be increased to allow specific amplification (the primershave more time interval to hybridize to target nucleic acids) inaddition to reduced or minimal primer dimers. The concentration oftarget-specific primers can be about 500 nM, 250 nM, 100 nM, 80 nM, 70nM, 50 nM, 30 nM, 10 nM, 2 nM, 1 nM or lower than 1 nM. Alternatively,the concentration of each target-specific primer can be between 1 μM to1 nM, between 1 nM to 80 nM, between 1 nM to 100 nM, between 10 nM to 50nM, or between 1 nM to 60 nM. The annealing temperature can be about 1minute, 3 minutes, 5 minutes, 8 minutes, 10 minutes or longer.Amplification with increased annealing times may use 1 cycle, 2 cycles,3 cycles, 5 cycles, 8 cycles, 10 cycles or more cycles followed bystandard annealing durations.

The disclosed methods and kits may comprise at least 10, 20, 100, 500,1,000, 2,500, 5,000, 10,000, 25,000, 50,000, 80,000, 100,000 or 150,000or more target-specific primers, wherein each target-specific primer isdirected to hybridize to a specific target sequence. There can be morethan one set of target-specific primers; as an example, there can be twosets of target-specific primers for two test reactions, 3 sets for 3test reactions or 5 sets for 5 test reactions or more. For practicalreasons, such as limitations in target-specific primer design orselection, the sample may also be split into multiple parallel multiplextest reactions with multiple sets of target-specific primers.

The GC content of target-specific primers can be between 40% to 70%,between 30% to 60%, between 50% to 80%, or between 30 to 80%.Alternatively, the target-specific primer GC content range can be less20%, 15%, 10% or 5%. The melting temperature (T_(m)) of thetarget-specific primers can be between 55° C. to 65° C., between 40° C.to 70° C., between 50° C. to 68° C., or such other range as determinedby one of skill in the art. The melting temperature range of thetarget-specific primers can vary. In some instances, the range can beless 20° C., 15° C., 10° C., 5° C., 2° C. or 1° C. The length of thetarget-specific primers can also vary. In some instances, the length canbe between 20 to 90 bases, 40 to 70 bases, 20 to 40 bases or 25 to 50bases. The range of length of the target-specific primers can also vary.For instance, it can be 60, 50, 40, 30, or 20 bases. In some instances,the 5′-region of the target-specific primer is an auxiliary or universalprimer binding site or a tag and is not complementary or specific forany target sequence. In some instances, the length of the targetsequence is between 50 and 500 bases, 90 to 350 bases, or 200 to 450bases, although other lengths are possible.

The present disclosure is also directed to a kit that comprises two ormore target-specific primers. In some instances, the kit comprises aplurality of methylated target-specific primers. In other instances, thekit comprises a combination of methylated universal primers andtarget-specific primers. The target-specific primers are designed andselected based on criteria described to have no or minimal primer-primerinteractions or non-specific priming. The kit can be formulated fordetection, diagnosis, prognosis and treatment of disease such as canceror congenital or inherited disease. The kit can also be configured forploidy status of a gestating fetus, for example by selectingtarget-specific primers that target sequences on chromosomes that areassociated with trisomy in fetus such as chromosomes 13, 18, 21, X andY, other chromosome, or some combination thereof. The kit may compriseabout 10, 20, 100, 500, 1,000, 2,500, 5,000, 10,000, 25,000, 50,000,80,000, 100,000 or 150,000 or greater target-specific primers.

The methods and kits disclosed herein may comprise a plurality oftarget-specific primers having no or minimal self-complementarystructure and that do not form a secondary structure, such as hairpinsor loops. The methods and kits disclosed herein may further comprise aplurality of target-specific primers having minimal cross-hybridizationto non-specific sequences present in a sample.

The target-specific primers disclosed herein may be used for efficientamplification of short nucleic acid fragments, such nucleic acidsderived from FFPE samples, cell free DNA (cfDNA), cell free tumor DNA(ctDNA) and cell free fetal DNA (cffDNA). The short nucleic acidfragments can be less than about 40 bases, 50 bases, 60 bases, 70 bases,80 bases, 90 bases, 100 bases or 120 bases. The method discloses hereinmay also be used for the detection and quantification of minoritymutations lower than 1%, such as T790M mutation related to drugresistance in lung cancer.

Methods disclosed herein produce an amplification product that can besequenced by next-generation sequencing platforms. Next-generationsequencing is referred to non-sanger based massively parallel DNAnucleic acid sequencing technologies that can sequence tens of thousandsof, or millions to billions of DNA strands in parallel. Examples ofcurrent state of state-of-art next-generation sequencing technologiesand platforms are Illumina platforms (reversible dye-terminatorsequencing), 454 pyrosequencing, Ion Semiconductor sequencing (IonTorrent), PacBio SMRT sequencing, Qiagen GeneReader sequencingtechnology, and Oxoford Nanopore sequencing. The present disclosure isnot limited to these next-generation sequencing technologies examples.In another aspect, the methods disclosed herein can be used in amultiplex fashion when amplifying more than two targets. The methodsdisclosed herein are not limited to any number of multiplexing.

EXAMPLES Example 1

Multiplex Amplification with Methylation-Modified Primers forIdentification of Variants (Approach 1)

Materials and Methods

Human DNA was extracted by Qiagen DNA extraction kit according to themanufacturer's instructions and the quantity of DNA was measured both byNanoDrop (ThermoFisher, USA) and Qubit 3 (ThermoFisher, USA).

Three oncogenes were selected for this experiment. Methylation-modifiedforward and reverse primers were designed for detection of variants forEGFR, KIT, and TP53 oncogenes. Each primer consisted of atarget-specific region and a methylation-modified auxiliary universalregion.

Multiplex PCR was performed applying 6 methylation-modified primers inpresence of genomic DNA, DNA polymerase, dNTP and PCR buffer in a 20 μlreaction volume. The PCR conditions consisted of initiation at 98° C.for 30 sec, 15 cycles of 98° C. 10 sec, 63° C. 4 min, 72° C. 20 sec andfinal extension at 72° C. 2 min.

Exonuclease I (NEB, USA) digestion to remove redundant primers andSPRIselect (Beckman Coulter, USA) beads purification to select largefragments was performed on the amplified product according to themanufacturer's instructions.

The PCR products were digested in separate reactions with either MspJIor LpnPI (NEB, USA) according to the manufacturer's instructions. Thedigested products were purified by SPRIselect (Beckman Coulter, USA)beads.

The digested products were ligated to adapters containing complementarysticky ends using Instant Sticky-end Ligase Master Mix (NEB, USA)according to manufacturer's instructions. The procedures were performedon an Applied Biosystems Veriti thermal cycler (ThermoFisher, USA).

The ligated DNA products were purified by SPRIselect (Beckman Coulter,USA) beads to remove surplus ligation adapters.

PCR was performed on ligated products using barcoded universal primers,hybridizing to ligated product universal priming site in presence of DNApolymerase, dNTP and PCR buffer in 20 μl reaction volume. The PCRconditions consisted of initiation at 98° C. for 30 sec, 21 cycles of98° C. 10 sec, 68° C. 30 sec, 72° C. 20 sec and final extension at 72°C. 2 min.

The amplified products was digested with Exonuclease I (NEB, USA) toremove redundant primers and was purified by SPRIselect beads (BeckmanCoulter, USA), and was then measured on a Qubit 3 (ThermoFisher, USA).The final products were sequenced bidirectionally by Sanger sequencing.

Example 2

Cancer Gene Panel for Identification of Mutations and Fusion Genes inLung Cancer from FFPE Samples

Materials and Methods

Human genomic DNA was used for this experiment to analyze possiblemutations that can affect the treatment regimen.

The DNA was extracted by Qiagen DNA extraction kit according to themanufacturer's instructions and the quantity of DNA was measured both byNanoDrop (ThermoFisher, USA) and Qubit 3 (ThermoFisher, USA).

Cancer gene and primer design: based on literature search, 15 cancerrelated genes were selected: AKT1, ALK, BRAF, CTNNB1, EGFR, ERBB2, HRAS,KIT, KRAS, MAP2K1, MET, NRAS, PDGFRA, PIK3CA and TP53. For detection ofhotspot mutations on these genes, 61 pairs of forward and reverseprimers were designed for multiplex amplification of target nucleicacids.

Multiplex PCR was performed applying 122 target-specific primerscontaining a universal auxiliary sequence in the presence of genomicDNA, DNA polymerase, dNTP and PCR buffer in a 20 μl reaction volume. ThePCR conditions consisted of initiation at 98° C. for 30 sec, 10 cyclesof 98° C. 10 sec, 63° C. 4 min, 72° C. 20 sec and final extension at 72°C. 2 min.

The amplified product from the first amplification was subjected toexonuclease I (NEB, USA) treatment to remove redundant primers accordingto the manufacturer's instructions. Then, the resulting first ampliconwas purified by SPRIselect beads (Beckman Coulter, USA).

Amplification was performed using a portion of the purified product fromthe first amplification and methylated universal auxiliary primers inthe presence of DNA polymerase, dNTP and PCR buffer in a 20 μl reactionvolume. The PCR conditions consisted of initiation at 98° C. for 30 sec,15 cycles of 98° C. 10 sec, 69° C. 30 sec, 72° C. 30 sec and finalextension at 72° C. 2 min.

Exonuclease I (NEB, USA) digestion to remove redundant primers andSPRIselect beads (Beckman Coulter, USA) purification to select largefragments was performed on the amplified product according to themanufacturer's instructions.

The amplified product was digested using either MspJI or LpnPI (NEB,USA) according to the manufacturer's instructions and was then purifiedby SPRIselect beads.

The digested products were ligated to adapters containing complementarysticky ends using Instant Sticky-end Ligase Master Mix (NEB, USA)according to manufacturer's instructions. The procedures were performedon an Applied Biosystems Veriti thermal cycler (ThermoFisher, USA).

The ligated DNA products were then purified by SPRIselect beads toremove surplus ligation adapters.

PCR was performed on ligated products using barcoded universal primers,hybridizing to ligated product universal priming site in presence of DNApolymerase, dNTP and PCR buffer in 20 μl reaction volume. The PCRconditions consisted of initiation at 98° C. for 30 sec, 21 cycles of98° C. 10 sec, 68° C. 30 sec, 72° C. 20 sec and final extension at 72°C. 2 min.

The amplified products was digested with Exonuclease I (NEB, USA) toremove redundant primers and was purified with SPRIselect beads, and wasthen measured on a Qubit 3 (ThermoFisher, USA).

Sequencing of the libraries were performed on a MiniSeq sequencingsystem (Illumina, CA, USA) using MiniSeq Mid Output Kit.

The sequence data generated from the above experiment was analyzed formutations and variations.

The methodologies and the various embodiments thereof described hereinare exemplary. Various other embodiments of the methodologies describedherein are possible.

Now, therefore, the following is claimed:
 1. A method of enrichingnucleic acid target sequences in a sample, comprising the steps of: in atest reaction, hybridizing two or more target-specific primers to targetsequences in the sample, wherein the target-specific primers comprise amethylated universal auxiliary portion with a methylation-dependentendonuclease restriction enzyme recognition site and a target-specificportion configured to target the nucleic acid target sequences in thesample; subjecting the test reaction to amplification under optimalamplification conditions to produce an amplified product comprising anamplicon; subjecting the amplified product to digestion with amethylation-dependent endonuclease restriction enzyme to form adigestion product, wherein the digestion product comprises ampliconscomprising sticky ends on each end of the strands; performing sizeselection purification on the digestion product for removal of digestedprimer-dimers and unused primers to produce digested ampliconscomprising dsDNA; ligating universal adapters to dsDNA from the digestedamplicons to form a ligation product, wherein the ligation universaladapters comprise a universal sequence portion and sticky ends; andsubjecting the ligation product to amplification with barcoded universalprimers complementary to a sequence on the ligating universal adaptersto form a final amplification product comprising final amplicons.
 2. Themethod of claim 1, further comprising at least one additional set oftarget-specific primers in at least one additional test reaction.
 3. Themethod of claim 1, wherein the sample comprises genomic DNA.
 4. Themethod of claim 1, wherein the sample comprises RNA and furthercomprising the step of, prior to first amplification, subjecting the RNAto a reverse transcription reaction to generate double-stranded cDNA. 5.The method of claim 1, wherein the method further comprises the step ofsubjecting the final amplicons to next-generation sequencing to generatesequence data.
 6. The method of claim 5, further comprising the step ofmeasuring allele counts at polymorphic sites in the sequence data. 7.The method of claim 1, wherein the ligation universal adapters furthercomprise a barcode sequence.
 8. The method of claim 1, wherein thesample comprises nucleic acid selected from the group consisting of: amixture of maternal cfDNA and cffDNA obtained from a pregnant subject,circulating cfDNA and circulating ctDNA.
 9. The method of claim 1,wherein the target-specific primers comprise at least one pair offorward target-specific primers and reverse target-specific primers. 10.The method of claim 1, wherein the target sequences comprise one or moremutations that are associated with disease, cancer, disorders,infections, pharmacogenetic drug treatment (companion diagnostic), drugresistance or drug antibiotic resistance, or aneuploidy or trisomy in agestating fetus.
 11. A method of enriching nucleic acid target sequencesin a sample, comprising the steps of: in a test reaction, hybridizingtwo or more target-specific primers to nucleic acid target sequences,wherein the target-specific primers comprise a complementary universalauxiliary portion at the 5′-end and a target-specific portion configuredto target the nucleic acid target sequences in the sample; subjectingthe test reaction to a first amplification with universal auxiliaryprimers under optimal amplification conditions to form an amplifiedproduct; subjecting a portion of the amplified product to a secondamplification using a methylated universal auxiliary primer to form asecond amplified product, wherein the methylated universal auxiliaryprimer comprises a restriction enzyme recognition sequence; subjectingthe second amplified product to digestion with a methylation-dependentendonuclease restriction enzyme to form a digestion product comprisingamplicons comprising sticky ends on each end of the strands; performingsize selection purification on the digestion product to remove digestedprimer-dimers and unused primers to form digested amplicons comprisingdsDNA; ligating universal adapters to dsDNA from the digested ampliconsto form a ligated product, wherein the ligating universal adaptorscomprise complementary sticky ends and a universal sequence portion; andsubjecting the ligated product to a third amplification using barcodeduniversal primers complementary to sequences on the ligating universaladapters to form a final amplification product comprising finalamplicons.
 12. The method of claim 11, further comprising at least oneadditional set of target-specific primers in at least one additionaltest reaction.
 13. The method of claim 11, wherein the sample comprisesgenomic DNA.
 14. The method of claim 11, wherein the sample comprisesRNA and wherein the method further comprises, prior to the firstamplification, subjecting the RNA to a reverse transcription reaction togenerate double-stranded cDNA.
 15. The method of claim 11, wherein themethod further comprises the step of subjecting the final amplicons tonext-generation sequencing to generate sequence data.
 16. The method ofclaim 15, further comprising the step of measuring allele counts atpolymorphic sites in the sequence data.
 17. The method of claim 11,wherein the ligation universal adapters further comprise a barcodesequence.
 18. The method of claim 11, wherein the sample comprisesnucleic acid selected from the group consisting of: a mixture ofmaternal cfDNA and cffDNA obtained from a pregnant subject, circulatingcfDNA and circulating ctDNA.
 19. The method of claim 11, wherein thetarget-specific primers comprise at least one pair of forwardtarget-specific primers and reverse target-specific primers.
 20. Themethod of claim 11, wherein the target sequences comprise one or moremutations that are associated with disease, cancer, disorders,infections, pharmacogenetic drug treatment (companion diagnostic), drugresistance or drug antibiotic resistance, or aneuploidy or trisomy in agestating fetus.